Zero-mode waveguides for single-molecule fluorescence imaging were fabricated using a simple desktop UV nanoimprint lithography system. An array of 30-to 150-nm-diameter nanoholes was successfully fabricated in an aluminum layer on a thin quartz plate by the single-step lift-off process using the UV-curable resist NIAC 707. Using the nanoholes, we performed real-time single-molecule fluorescence imaging to visualize the cochaperonin GroES binding with and dissociating from the chaperonin GroEL immobilized within the nanoholes. The demonstration revealed that the fluorescence from the GroES binding with the GroEL was three times stronger than the fluorescence from the GroES undergoing Brownian motion, and the real-time single-molecule fluorescence imaging was feasible using the zero-mode waveguide fabricated by the UV nanoimprint lithography lift-off process.
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